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Antiviral Research (v.72, #2)

Notes to Authors (pp. co3).

Editorial Board (pp. ii).

Announcement (pp. 77-77).

Understanding the accessory viral proteins unique to the severe acute respiratory syndrome (SARS) coronavirus by Yee-Joo Tan; Seng Gee Lim; Wanjin Hong (pp. 78-88).

A novel coronavirus, termed the severe acute respiratory syndrome coronavirus (SARS-CoV), infected humans in Guangdong, China, in November 2002 and the subsequent efficient human-to-human transmissions of this virus caused profound disturbances in over 30 countries worldwide in 2003. Eventually, this epidemic was controlled by isolation and there has been no human infection reported since January 2004. However, research on different aspects of the SARS-CoV is not waning, as it is not known if this virus will re-emerge, especially since its origins and potential reservoir(s) are unresolved. The SARS-CoV genome is nearly 30kb in length and contains 14 potential open reading frames (ORFs). Some of these ORFs encode for genes that are homologous to proteins found in all known coronaviruses, namely the replicase genes (ORFs 1a and 1b) and the four structural proteins: nucleocapsid, spike, membrane and envelope, and these proteins are expected to be essential for the replication of the virus. The remaining eight ORFs encodes for accessory proteins, varying in length from 39 to 274 amino acids, which are unique to SARS-CoV. This review will summarize the expeditious research on these accessory viral proteins in three major areas: (i) the detection of antibodies against accessory proteins in the serum of infected patients, (ii) the expression, processing and cellular localization of the accessory proteins, and (iii) the effects of the accessory proteins on cellular functions. These in-depth molecular and biochemical characterizations of the SARS-CoV accessory proteins, which have no homologues in other coronaviruses, may offer clues as to why the SARS-CoV causes such a severe and rapid attack in humans, while other coronaviruses that infect humans seem to be more forgiving.

Keywords: Severe acute respiratory syndrome (SARS); Coronavirus; Accessory viral proteins; Virus–virus interactions; Virus–host interactions

Inhibition of HIV-1 replication by a peptide dimerization inhibitor of HIV-1 protease by David A. Davis; Cara A. Brown; Kathleen E. Singer; Victoria Wang; Joshua Kaufman; Stephen J. Stahl; Paul Wingfield; Kenji Maeda; Shigeyoshi Harada; Kazuhisa Yoshimura; Pope Kosalaraksa; Hiroaki Mitsuya; Robert Yarchoan (pp. 89-99).

Peptides based on the amino (N) and carboxy (C)-terminal regions of human immunodeficiency virus type-1 (HIV-1) protease and on the C-terminus of p6^* can inhibit HIV-1 protease activity by preventing dimerization. We developed a peptide dimerization inhibitor, P27, that included these domains and a cell permeable domain derived from HIV-1 Tat. P27 inhibited wild type (WT) and protease inhibitor (PI)-resistant HIV-1 protease (IC₅₀: 0.23–0.32μM). Kinetic and biochemical assays confirmed that P27 inhibits protease dimerization. Fluorescein-labeled peptide accumulated in MT-2 cells and protected acutely infected MT-2 cells from HIV-1-induced cytotoxicity (IC₅₀: 5.1μM). P27 also inhibited p24 accumulation from H9 and U937 cells chronically infected with WT or PI-resistant HIV-1. Immunoblot analysis on the supernatants and infected cells revealed a block in virus release by P27 rather than an inhibition of polyprotein processing. However, inhibition of p55 Gag processing by active-site inhibitors was enhanced when combined with P27, suggesting that P27 can affect protease function in maturing virions. Although P27 was rationally designed to block dimerization of the mature HIV-1 protease, the effects of P27 on HIV-1 replication may be related to partial inhibition of Gag-Pol processing leading to a disruption in virus release.

Keywords: Abbreviations; HIV-1; human immunodeficiency virus type-1; WT; wild type; PI; protease inhibitor; CPD; cell permeable domain; CPP; cell permeable peptide; RP-HPLC; reverse phase high performance liquid chromatography; PBS; phosphate-buffered salineHIV-1; Protease; Dimer; Peptide; Inhibitor; Polyprotein

The flavonoid ellagic acid from a medicinal herb inhibits host immune tolerance induced by the hepatitis B virus-e antigen by Eun Hwa Kang; Tae Young Kown; Goo Taeg Oh; Weung Feel Park; Sung-Il Park; Sung Kyu Park; Young Ik Lee (pp. 100-106).

The aim of this study is to characterize the role of ellagic acid, a flavonoid from a medicinal herb which blocks HBeAg secretion in a HBV infected cell line and in HBeAg transgenic mice, in immune tolerance in chronic HBV infection. Using the mouse strain C57ML/6, HBeAg-producing transgenic mice (HBeAg-Tg), under the control of metal ion-inducible promoter were generated. The effect on immune tolerance of HBeAg-Tg and the release of immune tolerance by the inhibitor of HBeAg secretion, ellagic acid, was tested using T/B cell proliferation, HBeAg/HBeAb production, cytotoxic T-lymphocyte (CTL) and cytokine assays.C57ML/6 based HBeAg-producing HBeAg-Tg mice were tolerant to HBeAg at the T and B-cell level, did not produce antibodies to HBeAg in vivo and in vitro, produced minimal levels of cytokines (IL-4 and IFN-gamma) and decreased CTL responses, while feeding mice with ellagic acid (5mg/kg body weight) blocked the immune tolerance caused by HBeAg. Our results suggest that host immune tolerance induced by HBeAg during HBV infection, a viral strategy to guarantee HBV infection, can be overcome by ellagic acid, thus it can be used as a therapeutic for HBV-carriers.

Keywords: Hepatitis B virus–e antigen; Immune tolerance; Ellagic acid; Anti-HBV infection

Identification of selective inhibitors of VHSV from biased combinatorial libraries of N, N′-disubstituted 2,5-piperazinediones by V. Mas; A. Falco; I. Brocal; L. Perez; J.M. Coll; A. Estepa (pp. 107-115).

A combinatorial strategy has been used to design and identify inhibitors of viral haemorragic septicemia virus (VHSV), a salmonid rhabdovirus of economic importance. Two libraries of N, N′-disubstituted 2,5-piperazinediones (DKP), DKP-I and DKP-II were screened for inhibition of VHSV infectivity. Among the 98 DKP-derivatives (R1-DKP-R2) screened, a novel class of VHSV in vitro inhibitors was identified. Evidences are presented showing that the selected DKP-derivatives cause dose-dependent inhibition of VHSV infectivity in the absence of cellular toxicity. Preliminary characterization of its inhibition mechanism ruled out direct inactivation of the virus (virucidal effect) or interference with early viral replication steps. Furthermore, analysis of infection foci sizes, virus titers, viral protein accumulation and presence of cell free virus derived from VHSV-infected cell cultures in the presence of DKP-derivates suggested that virus assembly/release was impaired leading to a reduced virus spread in cell culture. New DKP-derivatives with a significant higher specific activity need to be developed to start testing its possible practical use but the selected DKP-derivatives described here may contribute to their further development as well as being tools to improve our knowledge on the fish rhabdovirus infection cycle.

Keywords: VHSV; Rhadovirus; Piperazinediones; Combinatorial libraries; Inhibitors; Antivirals

Hepatitis B virus e antigen production is dependent upon covalently closed circular (ccc) DNA in HepAD38 cell cultures and may serve as a cccDNA surrogate in antiviral screening assays by Tianlun Zhou; Haitao Guo; Ju-Tao Guo; Andrea Cuconati; Anand Mehta; Timothy M. Block (pp. 116-124).

Currently available antiviral nucleoside analogs for the treatment of chronic hepatitis B virus (HBV) infections profoundly reduce virus load, but rarely cure the virus infection. This is due, at least in part, to their failure to eliminate viral covalently closed circular (ccc) DNA from the nuclei of infected hepatocytes. To screen compound libraries for antiviral drugs targeting cccDNA, we set out to develop a cell-based assay suitable for high throughput screening. Since cccDNA is time-consuming to assay, it was desirable to use a viral gene product that could serve as a reporter for intracellular cccDNA level. We predicted that the secretion of HBV e antigen (HBeAg) by HepAD38 cells, a tetracycline inducible HBV expression cell line, would be cccDNA-dependent. This is because a large portion of pre-core mRNA leader sequence in the 5′ terminus of integrated viral genome was deleted, preventing HBeAg expression from transgene, but could be restored from the 3’ terminal redundancy of pre-genomic RNA during viral DNA replication and subsequent cccDNA formation. Our experimental results showed that following induction, HepAD38 produced and accumulated cccDNA, which became detectable between 7 and 8 days. HBeAg synthesis and secretion into culture fluid were dependent upon and proportional to the level of cccDNA detected. Therefore, the secretion of HBeAg by HepAD38 cells could potentially serve as a convenient reporter for the high throughput screening of novel antiviral drugs targeting HBV cccDNA.

Keywords: Hepatitis B virus; cccDNA; Antiviral assay

Effects of cidofovir treatment on cytokine induction in murine models of cowpox and vaccinia virus infection by Corinna W. Knorr; Stanley D. Allen; Anthony R. Torres; Donald F. Smee (pp. 125-133).

Cytokine profiles during cowpox and vaccinia (WR strain) virus infections were characterized in intranasal (i.n.) and intraperitoneal (i.p.) models in BALB/c mice. The time-course of induction and effects of cidofovir treatment on interferon (IFN)-γ, IFN-γ inducible protein (IP)-10, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1 were determined. The four mouse infection models have distinct patterns of cytokine induction. Cowpox virus i.p. and vaccinia virus i.n. infections showed increased induction throughout the time studied. Cowpox virus i.n. infection resulted in delayed induction of IFN-γ and IP-10. Cytokine levels were fairly constant during vaccinia virus i.p. infections. Cidofovir treatment (100mg/kg/day i.p. for 2 days) significantly suppressed certain cytokine (IFN- γ, IL-6, IL-10, IL-11, IP-10, LIF, MCP-1, MCP-3, MCP-5, MIP-1γ, and TIMP-1) levels to near normal relative to uninfected animals, as well as prevented mortality and reduced virus titers significantly. Characterization of cytokine responses has implications for understanding the immune responses and pathogeneses of viral infections in these mouse models.

Keywords: Orthopoxvirus; Cowpox; Vaccinia; Cidofovir; Antiviral; Cytokines

Potent inhibition of HIV-1 gene expression and TAT-mediated apoptosis in human T cells by novel mono- and multitarget anti-TAT/Rev/Env ribozymes and a general purpose RNA-cleaving DNA-enzyme by Hoshang Unwalla; Samitabh Chakraborti; Vikas Sood; Nidhi Gupta; Akhil C. Banerjea (pp. 134-144).

One of the hallmarks of progression of HIV-1/AIDS is the rapid depletion of CD4+T cells that is known to occur at the late stages of the disease when usually X4 tropic HIV-1 predominates. Besides direct killing of T lymphocytes, HIV-1 infection leads to extensive apoptosis of naïve/uninfected bystander T cells, which is predominantly mediated by HIV-1 TAT protein. Therefore, reduction of HIV-1 TAT protein is likely to reduce substantially the pathogenesis associated with HIV-1 infection. We designed two non-GUX hammerhead ribozymes (Rzs) and a Di-Rz by placing them in direct tandem. These were targeted against the most conserved second exon of HIV-1 TAT/Rev/Env region. Although very impressive in vitro cleavage of the target RNA by the two hammerhead Rzs was obtained under standard conditions of cleavage, only one of them was active under simulated physiological conditions. Sequence-specific cleavage by the Di-Rz was most efficient under standard conditions. Cleavage reactions carried out under simulated physiological conditions by the Di-Rz, however, indicated that both mono-Rzs were functional. We also assembled a 10–23 catalytic motif containing general purpose RNA-cleaving DNA-enzyme (Dz) against the same region, which cleaved the target RNA very efficiently. Both Rzs and Dz showed not only potent inhibition of HIV-1 gene expression but also showed remarkable protection against HIV-1 TAT protein-mediated apoptosis in Jurkat T cells. Possible implications of these findings are discussed.

Keywords: Abbreviations; Rz; ribozyme; Dz; DNA-enzyme; RE; restriction enzymeHIV; Catalytic nucleic acids; Apoptosis; Gene therapy

Lactoferrin inhibits early steps of human BK polyomavirus infection by Giovanna Longhi; Valeria Pietropaolo; Monica Mischitelli; Catia Longhi; Maria Pia Conte; Magda Marchetti; Antonella Tinari; Piera Valenti; Anna Marta Degener; Lucilla Seganti; Fabiana Superti (pp. 145-152).

Lactoferrin, a member of the transferrin family, is a bi-globular iron binding glycoprotein, found in milk, exocrine secretions of mammals, and in secondary granules of polymorphonuclear neutrophiles that plays an important role in the defence against various pathogenic microorganisms. Previous studies in different virus-cell systems showed that lactoferrin is a potent inhibitor of different enveloped and naked virus infection. In this research we studied the effect of lactoferrin on BK polyomavirus, a human naked double-stranded DNA virus responsible for productive, persistent, and latent infections of the urinary tract. Results obtained demonstrate that lactoferrin treatment prevents early steps of BK virus infection in Vero cells, at the level of the adsorption phase, probably through the interaction with capsidic structures, although a lactoferrin-BK virus competition for cell plasma-membrane receptors cannot be ruled out.

Keywords: BK virus; Lactoferrin; Antiviral agents

In vitro and in vivo anti-retroviral activity of the substance purified from the aqueous extract of Chelidonium majus L. by Marijan Geren�?er; Peter L. Turecek; Otfried Kistner; Artur Mitterer; Helga Savidis-Dacho; Noel P. Barrett (pp. 153-156).

We have isolated a substance with anti-retroviral activity from the freshly prepared crude extract of Chelidonium majus L. (greater celandine) by 9-aminoacridine precipitation method and ion exchange chromatography using Dowex-50W/H+ resin followed by the gel filtration on Sephadex-75 column. Elemental and phenol/sulfuric acid method analyses as well as the mass spectrometry of the purified substance indicated that it may represent a low-sulfated poly-glycosaminoglycan moiety with molecular weight of ∼3800Da. The substance prevented infection of human CD4+ T-cell lines AA2 and H9 with HIV-1 at concentration of 25μg/mL as well as the cell-to-cell virus spread in H9 cells continuously infected with HIV-1, as determined by the measurement of reverse transcriptase activity and p24 content in cell cultures. Furthermore, we have shown in a murine AIDS model that the treatment with purified substance significantly prevented splenomegaly and the enlargement of cervical lymph nodes in C57Bl/6 mice chronically infected with the pool of murine leukemia retroviruses. The mechanism(s) of anti-retroviral activity of this substance have to be elucidated.

Keywords: Chelidonium; Glycosaminoglycans; HIV-1 inhibition; Retroviruses; MAIDS

Synergistic antiviral activity of acyclovir and vidarabine against herpes simplex virus types 1 and 2 and varicella-zoster virus by Mikiko Suzuki; Tomoko Okuda; Kimiyasu Shiraki (pp. 157-161).

Acyclovir and vidarabine both exhibit anti-herpetic activity. Because different mechanisms of action of vidarabine and acyclovir have been reported, we analyzed their combined anti-herpetic activity on plaque formation of herpes simplex virus (HSV)-1, HSV-2, and varicella-zoster virus (VZV) by isobolograms. The results indicate that acyclovir and vidarabine have a synergistic effect on wild type HSV-1, HSV-2, and VZV. The susceptibility of thymidine kinase-deficient HSV-1 to vidarabine was not affected by the presence of acyclovir, suggesting that phosphorylation of acyclovir is essential for synergism. The combined anti-HSV activity of acyclovir and vidarabine against phosphonoacetic acid-resistant HSV-1 with DNA polymerase mutation did not show synergism in contrast to that against wild-type herpesviruses. Alteration of the substrate specificity of viral DNA polymerase to acyclovir and vidarabine annihilated the synergism. Thus, the nature of their binding sites on DNA polymerase is important to the synergistic anti-herpesvirus activity of acyclovir and vidarabine.

Keywords: Herpes simplex viruses; Varicella-zoster virus; Drug interactions; Synergism; Acyclovir; Vidarabine

Inhibition of hepatitis B virus by D-fraction from Grifola frondosa: Synergistic effect of combination with interferon-α in HepG2 2.2.15 by Chang-Qing Gu; Jun-Wen Li; Fu-Huan Chao (pp. 162-165).

In this study, D-fraction extracted from Grifola frondosa (GF-D) and its combination with human interferon alpha-2b (IFN) were investigated for the inhibitory effect on hepatitis B virus (HBV) in HepG2 2.2.15 cells (2.2.15 cells). HBV DNA and viral antigens were analyzed by a quantitative real-time polymerase chain reaction and end-point titration in radioimmunoassays, respectively. The results showed that GF-D or IFN alone could inhibit HBV DNA in 2.2.15 cells with the 50% inhibitory concentration (IC₅₀) of 0.59mg/ml and 1399IU/ml, respectively. We further investigated the combination of GF-D and IFN for anti-HBV activity and found that they synergistically inhibited HBV replication in 2.2.15 cells. In combination with 0.45mg/ml GF-D, the apparent IC₅₀ value for IFN was 154IU/ml. This 9-fold increase in antiviral activity of IFN suggested that GF-D could synergize with IFN. These results indicate that GF-D, in combination with IFN, might provide a potentially effective therapy against chronic HBV infections.

Keywords: Antiviral; Grifola frondosa; Interferon; Hepatitis B virus; Combination

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Antiviral Research (v.72, #2)